Heterogenous and homogeneous mouse variants lacking sno gene

ABSTRACT

Functions of sno gene are analyzed by constructing heterogenous or homogeneous mouse variants lacking sno gene to thereby provide heterogenous or homogeneous mouse variants lacking sno gene, cells of these animals and screening systems with the use of these animals or cells. Non-human animals, preferably mice, lacking sno gene and cells thereof. The above animals or cells thereof include both of heterogenous variants and homogeneous variants. A method for screening candidates for drugs by using the above animals or cells thereof.

TECHNICAL FIELD

The present invention relates to non-human animals, preferably mice,lacking the sno gene and cells thereof, preferably embryos thereof, or amethod for screening substances by using the above animals or cellsthereof.

BACKGROUND ART

A coactivator such as CBP, and a corepressor such as N-CoR, are calledas “mediating factors”. The mediating factors were identified asmolecule which acted as a bridge between the transcriptional regulatoryfactor binding to the enhancer/silencer upstream of the promoter and abasal transcription factor, such as TBP, binding to the core promoter.The mediating factors form complexes with histone acetyltransferase(HAT, an enzyme acetylating histone) or, on the contrary, with histonedeacetylase (HDAC, a deacetylating enzyme from histone), indicating thatit changes the chromatin structure through acetylation of histone inorder to regulate gene expression. The regulation of gene expression bymodifying the chromatin structure is thought to be involved in variouslife processes such as immunity and development and differentiation, butthe mechanisms are unknown.

Ikaros, which was originally thought to be a T cell specifictranscriptional activation factor, has been demonstrated, by recentstudies of a British group, to recruit a T cell specific gene into theheterochromatin region, which is the transcription inactivated region.It then plays a role in the expression of these genes inundifferentiated cells. As can be understood from this example, geneexpression regulation mediated by the chromatin structure is veryimportant in expression and differentiation of the immune system.Consequently, studies on the mediating factors provide a breakthrough inthis area.

One of the important matters in discussing the physiological function ofthe mediating factors is “haploinsufficiency”, which means that half isinsufficient. Since eucaryotic cells are diploid and normally have twocopies of genes, even if a mutation occurs in one copy of the gene, thephenotype does not appear in most cells. Studies by the inventors of thepresent invention have found that, in the case of the mediating factors,if a mutation occurs in one copy of the gene, the amount of gene productreduces to half. This process connects directly with an abnormalphenotype.

This leads to the idea that, since limited types and numbers of themediating factors are commonly used for large numbers of thetranscriptional regulatory factors, the reduction in the amount of geneproduct to half is directly related to the abnormal phenotype.Consequently, diseases which develop as a result of a mutation in themediating factors gene commonly occur in the group of so-called“autosomal dominant diseases (which mean onset occurs as a result of amutation of one copy of the gene in an autosome)”, and they frequentlyresult in a disease.

N-CoR was originally identified as a corepressor which is essential fortranscriptional regulation of the action of the intranuclear hormonereceptor and, together with a factor such as Sin3 or HDAC, constitutes alarge complex. We have analyzed the physiological function of ski/sno, aconstitutional factor of the N-CoR corepressor complex.

The ski gene was originally found as an oncogene which transforms thechicken embryonic fibroblastoma cells (Stavnezer, E., et al., Mol. CellBiol., 9, 4038-4945, 1989). We have found that the protein encoded bythe c-ski gene and its related gene sno (Nomura, N., et al., NucleicAcid Res., 17, 5489-5500, 1989) bind to the corepressor N-CoR/SMRT(Hoerlein, A. J., et al., Nature, 377, 397-404, 1995; Chen, J. D., etal., Nature, 377, 454-457, 1995), and to mSin3 (Ayer, D. E., et al.,Cell, 80, 767-776, 1995; Screiber-Agus, N., et al., Cell, 80, 777-786,1995), and form a complex with histone deacetylase (HDAC).

Ski/Sno is essential for the transcriptional repression which ismediated by thyroid hormone receptor and Mad. Further, these aredirectly bound with Rb as well as requiring an Rb-mediating repressivefactor.

We have found that Ski, which was identified as an oncogene product, andits related gene product Sno, bind directly with N-CoR and Sin3 tofunction as the constitutional factor of the N-CoR complex. Quiteinterestingly, the transcriptional suppression generated by the tumorsuppressive gene products Mad and Rb require Ski/Sno. Heterogeneous micelacking Sno were prepared and a carcinogenesis experiment was conducted.As a result, we have found that these mice were highly cancer-prone, andit was shown for the first time that the Ski/Sno gene family was a tumorsuppressor gene. In addition, heterogeneous mice lacking sno exhibitvarious abnormalities in their immune systems. The mice haveextraordinarily high levels of induced Th1 group cytokines such asinterferon γ, and easily develop ulcerative colitis. The germinal centerdoes not form in these mice and induction of B cell proliferation by LPSis not observed. We have also examined the relationships between variousabnormalities and diseases observed in mice in which the amount of Skior Sno is reduced to half the normal level, and their molecularmechanisms.

An object of the present invention is to provide heterogeneous orhomogeneous mouse variants lacking the sno gene and cells of theseanimals by constructing heterogeneous or homogeneous mouse variantslacking sno gene and analyzing functions of sno gene.

Another object of the present invention is to provide screening systemsinvolving the use of these animals or their cells.

DISCLOSURE OF THE INVENTION

The present invention includes non-human animals, preferably mice,lacking sno genes and cells thereof. The animals lacking sno gene andcells thereof of the present invention include both heterogeneousvariants and homogeneous variants.

Further, the present invention relates to a method for screeningcandidates for drugs by using the above animals or cells thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B: (a) shows restriction maps of vector and allele geneused in the present invention. (b) shows photographs instead of drawingsshowing results of analyses confirming expression in each genotype.

FIGS. 2: Photographs instead of drawings showing results of immunostainin each genotype.

FIGS. 3A and 3B: (a) shows a graph showing growth of cells in eachgenotype. (b) shows numerical indication of colony formations of eachgenotype in methylcellulose gel after infection with oncogenic virus.

FIGS. 4A-H: (a) a graph showing survival numbers of each genotype afteradministration of carcinogen DMBA. (b) a photograph instead of a drawingshowing lymphoma of mice developed tumor. (c) a photograph instead of adrawing showing metastasis to thymus. (d) a photograph instead of adrawing showing anti-TCR β (T cell receptor α and β) positive tumor. (e)a photograph instead of a drawing showing that tumor is not positive forB-cell marker IgM. (f) a photograph instead of a drawing showing tumorof pancreas of mice developed tumor. (g) and (h) photographs instead ofdrawings showing results of immunostain of mouse B lymphoma using T-celland B-cell specific antibodies.

FIGS. 5A and 5B: (a) shows loci in chromosome of human sno. (b)photographs instead of drawings showing results of assays in expressionof m-RNA of sno gene of three type cells, i.e. Saos-2, HG63 and OST.

BEST MODE FOR CARRYING OUT THE INVENTION

The method for preparing non-human animals lacking the gene of thepresent invention is described using mice as an example.

The sno mutant mice were developed by homologous recombination ofembryonic stem cells (ES cells).

A gene-targeting vector in the exon region encoding amino acids 1-362 ofSnoN, which is one of the multiple Sno protein species generated byalternative splicing, was replaced by a neo cassette [refer to FIG.1(a)]. Homologous recombinants were characterized by the appearance of a3.3 kb EcoO1091 fragment using the 5′ probe, and a 14.4 kb BglI fragmentusing the 3′ probe [refer to FIGS. 1(a) and (b)]. The chimeras werenormally obtained from two independent mutant ES clones.

The chimeras were mated with C57BL/6 or BALB/c females in order togenerate F1 heterozygous mutant mice. Heterozygous mice were identifiedby PCR and Southern blot analyses of genomic DNA isolated from tailsamples of the offspring [refer to FIG. 1(b)]. Heterozygous mice werecrossed, and the genotypes of the resulting 3 week old offspring weredetermined. Among 211 offsprings derived from two independent germ-linechimeras, no sno^(−/−) homozygotes were detected and the ratio of thesno^(+/+) to the sno^(+/−) genotype was 1:2. The fact that the ratio ofthe sno^(+/+) to the sno^(+/−) genotype in the progeny population was1:1 in the cross of the sno^(+/−) and the sno^(+/+) (either female ormale), indicated that the sno germ cells were not eliminated duringspermatogenesis or oogenesis.

To identify the time at which the sno^(−/−) animals died,postimplantation embryos (E3.5-E16.5) from heterozygous matings weresurgically explanted from the uterine tissue of pregnant females andtheir genotype was identified by PCR. No sno^(−/−) embryos were obtainedafter E3.5.

To characterize the preimplantation development of the sno^(−/−)embryos, 2-cell embryos derived from heterozygous xenogeneic crosses atE1.5 were recovered and cultured individually. Their development wasmonitored every 12 to 24 hours. The developmental potential of eachembryo was correlated with the presence of the Sno protein. The presenceof Sno protein was scored by immunostaining with Sno-specificantibodies. About 25% of the embryos (n=20) exhibited no significantstaining. All of these had a defect in blastocyst formation (refer toFIG. 2, right column). These embryos cleaved normally to the 8-cellstage during the first 24 hours in culture.

However, they became decompacted during the second and third days inculture, and failed to divide beyond approximately the 16-cell stage orto form a blastocoel. The remaining embryos were strongly labeled forSno and developed into normal blastocysts (refer to FIG. 2, leftcolumn). These results indicate that sno is required for blastocystformation. Since ski-deficient mice were alive even at E18.5 (Berk, M.,et al., Genes Dev., 11, 2029-2039, 1997), ski and sno have differentroles during development.

Ski overexpression in cultured cells is accompanied by growth inhibition(Colmenares, C., et al., Cell, 59, 293-303, 1989). In addition, normalc-Ski and Sno are both required for transcriptional repression by Madand Rb which negatively regulate cellular proliferation. To investigatethe possibility that sno negatively regulates cellular proliferation, westudied the growth of early passages of mouse embryonic fibroblasts(MEFs) prepared from E15.5 embryos, which express sno normally.

Although sno^(+/+) and sno^(+/−) cultures were morphologicallyindistinguishable at low density, sno^(+/−) MEFs grew faster thanwild-type MEFs [refer to FIG. 3(a)]. In addition, sno^(+/−) MEFmonolayers achieved higher cellular densities [refer to FIG. 3(a)]. Todetermine the basis for their increased proliferating capacity, weassayed the cell-cycle profile of wild type and sno^(+/−) MEFs.

In the sno^(+/−) samples, we observed an increased expression during theG₁ phase of the cell cycle which was proportional to the increase inproliferating capacity (data not shown). In order to further study theability to complete full transformation, colony formation inmethylcellulose was examined. After infection with a virus carrying thev-K-RAS oncogene, the sno^(+/−) MEFs generated 32-85 colonies per 106cells, whereas wild-type MEF generated no colonies at all [refer to FIG.3(c)].

Thus the loss of one copy of the sno gene allowed the activation of,ras-mediated transformation in cell culture.

In the next experiment, in order to compare the tumor susceptibility ofwild type and sno^(+/−) mice, the carcinogen9,10-dimethyl-1,2-benzanthracene (DMBA) was administered every week.Over the observation period of 80 days, most of the wild-type mice(n=40) survived even this treatment, but only 30% of the sno^(+/−)female mice (n=20) and 50% of the sno^(+/−) male mice (n=20) survivedthis treatment, The remaining mice produced clinically apparent tumors[refer to FIG. 4(a), p=0.004]. The major tumor type observed wasmalignant lymphomas, and fibromas were also found at a lower frequency.Lymphomas in sno^(+/−) mice were aggressive metastatic malignancies, andgenerated tumors in the thymus glands [refer to FIGS. 4(b) and (c)]. Thefact that these originated from T-cells is due to the tumor cellshomogeneously expressing the TCRαβ markers, but not the IgM marker[refer to FIGS. 4(d) and (e)].

The sno^(+/−) heterozygotes were monitored for the development of tumorsin the absence of carcinogen treatment. A predisposition for thedevelopment of tumorigenesis was observed to occur at a low frequency inthese heterozygotes. One mouse died at the age of 4 months afterdisplaying marasmoid symptoms. Histological analyses of various tissuesof this mouse revealed the presence of tumors in the pancreas [refer toFIG. 4(f)]. Immunostaining using T-cell and B-cell specific antibodiesindicated that the tumors were B lymphomas [refer to FIGS. 4(g) and(h)]. Generation of lymphomas is very consistent with the fact thatintroduction of a c-ski expression vector correlates withdifferentiation of hematopoietic cells (Namciu, S., et al,. Oncogene, 9,1407-1416, 1994), and the fact that v-ski can affect the growth of avianhematopoietic cells (Larsen, J., et al., Oncogene, 8, 3221-3228, 1993).

To investigate the possibility that the human sno locus is included inthe locus of tumor suppression, the location of the sno gene on thehuman chromosome was determined by using a GeneBridge 4 radiation-hybridpanel. As a result, the sno gene was localized between the WI-3847 andthe WI-6894 markers at 3q26.31-3q36.32 [refer to FIG. 5(a)]. Thedistance between these two markers can be estimated to be about 1500 kb.Quite interestingly, one of the highest frequencies of deficiency ofconstitutional heterozygosity for human osteosarcomas is also mapped inthis region, between the D3S 1212 and the D3S 1246 markers (Kruzelock,R. P., et al., Cancer Res., 57, 106-109, 1997)[refer to FIG. 5(a)].

These two regions containing the sno gene and the tumor suppressor forosteosarcomas overlap each other.

In order to determine whether or not sno expression is lost in cellsderived from osteosarcomas, sno mRNA expression in three proliferativecell lines, Saos-2, HG63 and OST was studied. The sno mRNA was notdetected in Saos-2 cells [refer to FIG. 5(b), left panel], but the othertwo cell lines expressed it at comparable levels to those found infibroblast cell lines. Southern blot analysis indicated that there wasno gross rearrangement in the sno gene in Saos-2 cells [refer to FIG.5(b), right panel], suggesting that the loss of sno expression may bedue to a small deletion, a rearrangement in a narrow region, or a pointmutation. It is well known that homozygous alterations occur in sporadicosteosarcomas involving both of the two best characterized tumorsuppressor genes, p53 and Rb 1, and that Saos-2 lacks the functional Rbprotein (Pompetti, F., et al., J. Cell. Biochem., 63, 37-50, 1996).

Although ski and sno have been thought to be oncogenes, so far theresults described above demonstrate that these genes act as tumorsuppressor. v-Ski lacks the C-terminal region of c-Ski to which mSin3binds, suggesting that v-Ski acts as a dominant negative form. Theoverexpression of not only v-ski but also c-ski transforms chickenembryonic fibroblasts (Colmenares, C., et al., J. Virol., 65, 4929-4935,1991).

We observe that overexpression of c-Ski and Sno are required fortranscriptional repression mediated by the proteins encoded by the twodifferent tumor suppressor genes, Rb and mad (Ayer, D. E., et al., GenesDev., 7, 2110-2119, 1993; Schreiber-Agus, N., et al., Nature, 393,483-487, 1998). We also note that the transcriptional repressor activityof these proteins may be decreased in sno heterozygous mutants, and theincreased expression of a group of target genes of Rb and Mad couldcause a predisposition to tumors.

We have recently found that the loss of one copy of the ski gene in MEFsincreases the predisposition to mutation, and that the human c-ski geneis localized at 1p36 (results are not shown here), where the multipletumor suppressor genes for neuroblastoma were also localized (Cheng, N.C., et al., Oncogene, 10, 291-297.), suggesting that c-ski is alsoestimated to be important for tumor susceptibility. Our finding that snoacts as a tumor suppressor gives a clue to understanding thephysiological role of the ski/sno gene family.

EXAMPLES

Following examples illustrate the present invention more concretely, butthe present invention is not limited within these examples.

Example 1 Generation of Sno Deficient Mutant Mice

The sno genomic clones were isolated from a library derived from TT2cells by the standard plaque hybridization procedure.

A 15.0 kb HindIII-EcoRI fragment containing the exon encoding theN-terminal xxx-amino acids region of Sno was deleted from theHindIII-EcoRI fragment and replaced with a neomycin (neo) cassettederived by using the phospholipid kinase gene promoter.

To increase the frequency of gene targeting, the DT-A [diphtheriatoxin-poly(A) signal] cassette for negative selection was fused to theshort arm (Yagi, T. et al., Anal. Biochem., 214, 77-86, 1993).

The ES cells used were TT2 cells derived from the F1 embryo resultingfrom a crossbreed between C57BL/6 and CBA mice (Yagi, T. et al., Anal.Biochem., 214, 70-76, 1993). Isolation of ES clones containing snomutation, generation of chimeras and production of heterozygous mutantswere performed as described previously (Tanaka, Y., et al., Proc. Natl.Acad. Sci. USA, 94, 10215-10220, 1997). The homologous nature of therecombination was confirmed by Southern blot analysis using severalrestriction enzymes and several probes located either inside or outsidethe targeting vector as described hereinbefore. Three different primers,shown in FIG. 1(a), were used to amplify a 240 bp fragment from the wildtype allele or a 120 bp fragment from the mutant allele. The mice weremaintained and bred in the Division of Experimental Animal Research,Physical and Chemical Research Institute, RIKEN, Japan.

Example 2 Detection of Sno Proteins

Splenocytes were washed with PBS and resuspended in xml of lysis bufferconsisting of 45 mM Tris-HCl (pH 7.4), 135 mM NaCl, 0.9% Triton X-100,0.9% sodium deoxycholate, 0.09% SDS, 22 mM EDTA, and 1% Trasylol. Aftercentrifugation, to xml of lysates were mixed with xml of NET/NP40 buffer[50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.5% NP40, and 1 mg/ml BSA]containing 150 mM NaCl. xml of 100 mM of PMSF and xml of ProteinG-Sepharose were added thereto, and the lysates were allowed to stand onice for 15 minutes, and centrifuged. The supernatant was mixed with theSno-specific monoclonal antibodies (5 mg of IgG) and the mixture wasallowed to stand on ice for 2 hours. The immuno-complex was collectedusing 20 ml of protein G-Sepharose, washed with NET/NP40 buffercontaining 0.5 and 0.25 M NaCl, NET buffer [50 Tris-HCl (pH 7.5) and 5mM EDTA] containing 0.15 M NaCl sequentially, and developed on a 10%SDS-PAGE gel. Proteins were transferred and separated on thenitrocellulose filter, and Sno was detected using the anti-Snomonoclonal antibodies (Tokitou, F., et al., J. Biol. Chem., 274,4425-4488, 1999) and ECL detection reagents (Amersham).

Example 3 In Vitro Culture of 2-cell Embryos and Immunostaining

M2 and M16 media were prepared (Hogan, B., et al., “Manipulating theMouse Embryo: A Laboratory Manual.” Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.). Embryos were obtained from matings betweenheterozygous sno^(+/−) males and females. The day a vaginal plug wasfound was defined as 0 day after postcoitum (dpc). Embryos were flushedout from the oviducts at 1.5 dpc using M2 medium. The embryos werecultured, individually in drops of M16 medium under paraffin oil at 37°C. in a humid atmosphere containing 5% CO₂. The embryos were examinedperiodically with a dissecting microscope. For immunostaining, embryoswere fixed at room temperature for 30 minutes in freshly prepared salinebuffer (pH 7.5) containing 2.5% paraformaldehyde, followed by treatmentwith methanol for 30 minutes and with 4% hydrogen peroxide for 30minutes. The primary antibody-antigen complexes were detected byperoxidase conjugated anti-mouse IgG (Dako) using anti-Sno monoclonalantibodies.

Example 4 Histological Analysis and Immunohistochemistry

Various tissues were fixed in 4% paraformaldehyde, dehydrated andembedded in paraffin. Sections (5 μm) were stained withhematoxylin-eosin staining according to the standard procedures.Paraffin sections cut at 4 μm thickness were used forimmunohistochemical examination. Anti-T cell receptor α and β, anti-IgM,anti-B220, and anti-CD3 antibodies were used for immunostaining.

Example 5 Analysis of Sno+/− Embryonic Fibroblasts and Splenocytes

Mouse primary embryonic fibroblasts were isolated from embryos at 13.5or 15.5 dpc. For growth curve experiments, 10⁵ cells per well wereseeded in six-well plates in DMEM containing 20% fetal bovine serum andcounted. Nine independent experiments were carried out from threedifferent MEF preparations. MEF cells were infected with Kirsten murinesarcoma virus (Ko-MuSV) and transfected with the activated K-rasoncogene (moi=10) for 48 hours. Cells (10⁵ cells) were suspended with1.3% methylcellulose gel and dissolved in culture medium and overlaid onan agarose bed composed of 0.53 agarose and culture medium. Colonieswere counted for scoring 3 weeks after plating.

Example 6 Treatment with Carcinogen, 9,10-dimethyl-1,2-benzanthracene(DMBA)

DMBA treatments were initiated from the beginning at postnatal day 2-5and continuing weekly thereafter by administering 50 μl of a solutioncontaining DMBA 1.25 mg per ml acetone to the dorsal surface. Mice weremonitored regularly and killed when overall health condition wasdeteriorated.

At first, 8-10 mice in each cohort were used for collectinghistopathological informations, subsequently, the site of tumor and thesite of metastatic organ were detected and specified by detailedpathological analysis.

INDUSTRIAL APPLICABILITY

The aspect of the present invention is to provide a strong clue for thescreening of carcinogens, and for elucidating the mechanisms of onset orsuppression of tumors. This can be done by identifying the new functionof the sno gene and by developing animals lacking the sno gene andconsequently having carcinogenic susceptibilities. An additional aspectof the present invention is to provide new methods for prevention andtreatment of tumors by using the sno gene or complementary sequencesthereof.

What is claimed is:
 1. A heterozygous knockout mouse comprising adisruption in the genomic DNA fragment encoding the N-terminal aminoacids of the sno gene and exhibiting one or more of tumorigenesis andpropensity to ulcerative colitis, wherein the HindIII-EcoRI fragment ofthe sno gene exon region is replaced with a neomycin gene under thecontrol of phospholipid kinase gene promoter.
 2. A knockout mouse cellcomprising a heterozygous disruption in the genomic DNA fragmentencoding the N-terminal amino acids of the sno gene and exhibiting oneor more of tumorigenesis and propensity to ulcerative colitis, whereinthe HindIII-EcoRI fragment of the sno gene exon region is replaced witha neomycin gene under the control of phospholipid kinase gene promoter.3. A cell according to claim 2, wherein the cell is isolated from anembryo.
 4. A method for screening drug candidates comprisingadministering to the mouse of claim 1, a candidate drug and assessingthe effects of the administration.
 5. A method for screening drugcandidates comprising administering to the cell of claim 2, a candidatedrug and assessing the effects of the administration.